Requirements for successful implementation of antisense therapeutic molecules, which are generally designed to bind to DNA or RNA of disease-causing proteins to prevent the production of such proteins, include (a) stability in vivo, (b) sufficient membrane permeability and cellular uptake, and (c) a good balance of binding affinity and sequence specificity. Many oligonucleotide analogs have been developed in which the phosphodiester linkages of native DNA are replaced by other linkages that are resistant to nuclease degradation (see e.g. Barawkar and Bruice 1998; Linkletter, Szabo et al. 2001; Micklefield 2001). Antisense oligonucleotides having various backbone modifications other than to the internucleoside linkage have also been prepared (Crooke 2001; Micklefield 2001). In addition, oligonucleotides have been modified by peptide conjugation in order to enhance cellular uptake (Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005).
The performance of such nucleic acid analogs as antisense or antigene drugs has been hampered by certain characteristics of the various analogs. For example, analogs with negatively charged linkages, including phosphorothioate-linked analogs, suffer from considerable electrostatic repulsion between the negative charges of the oligomer and the DNA or RNA target. The phosphorothioates also exhibit non-specific binding to other cellular components such as proteins. These attributes limit the usefulness of antisense oligomers comprised of native RNA, native DNA, and negatively charged analogs as therapeutic agents (Crooke 2001). The nonionic methylphosphonate-linked oligonucleotide analogs can be transported into cells by passive diffusion and/or fluid phase endocytosis, but their use is hampered by stereoisomeric complexity and poor solubility (Crooke 2001; Micklefield 2001).
Several groups have reported the synthesis of positively charged oligonucleotides (Bailey, Dagle et al. 1998; Micklefield 2001; Egli, Minasov et al. 2005). For example, a class of guanidinium linked nucleosides (designated DNG), formed by replacement of the phosphate linkages in DNA and RNA by achiral guanidino groups, has been reported (Dempcy, Almarsson et al. 1994; Dempcy, Luo et al. 1996; Barawkar and Bruice 1998; Linkletter, Szabo et al. 2001). Oligomers linked with positively charged methylated thiourea linkages have also been reported (Arya and Bruice 1999). Replacement of some of these linkages with neutral urea linkages is reported to reduce the tendency of such positively charged oligomers towards non-sequence-specific binding (Linkletter and Bruice, 2000). However, there remains a need for oligonucleotide analogs with improved antisense or antigene performance, particularly in the area of stronger affinity for DNA and RNA, without compromising sequence selectivity.